A robust method for ecigarette FDA-CTP PMTA guidance analytes: formaldehyde, acetaldehyde, acrolein, crotonaldehyde This presentation also includes details related to method optimization experiments. 2020_ST03_ZhuJ copy.pdf

1. Method Development for the Analysis of Mono-
Carbonyl Compounds in E-Vapor Products by LC-MS
ZHU J.*; HEREDIA A.; TWEEDY J.; TAYYARAH R.
ITG Brands, LLC, Greensboro, NC, USA
CORESTA 2020
*Email: jeff.zhu@itgbrands.com
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2. Introduction
• Mono-carbonyls (formaldehyde, acetaldehyde, acrolein and crotonaldehyde)
may be present, typically at trace levels, in e-vapor products.
• The traditional approach to analyze mono-carbonyls in cigarette smoke is to
derivatize with DNPH and analyze by HPLC-UV (Ref 1).
• However, UV detection is not suitable for carbonyls analysis in e-vapor products
because of lower selectivity and sensitivity as compared to mass spec
detection.
• We presented a new method on e-cigarette aerosols analysis by UPLC-MS with
good sensitivity and selectivity at TSRC 2019 (Ref 2 and references listed there
on recent development by GC-MS and LC-MS).
• We present now the steps we have taken during the method development
process to optimize the method and expand the scope to include e-liquids.
1) CORESTA Recommended Method No. 74 – Determination of Selected Carbonyls in Mainstream Cigarette
Smoke by HPLC, last updated in August 2019.
2) Zhu, J., Heredia, A., “A Simplified Method for The Analysis of Mono-Carbonyl Compounds in E-Cigarette Aerosols
By LC-MS”. 73rd Tobacco Science Research Conference, Sept. 2019, Leesburg, VA, USA.
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3. Optimization – Sample Preparation
pH Condition for Derivatization of Free Carbonyls
Optimal condition: 6 mM DNPH·HCl in water/acetonitrile 1:1
Total Trizma: 0.15 mL of 3.9 % Trizma for 7 mL of 6 mM DNPH·HCl solution.
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4. Optimization – Sample Preparation
- DNPH concentrations
Optimal condition: 6 mM DNPH·HCl in water/acetonitrile 1:1
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5. Summary for Sample Preparation
- Aerosol
• Aerosol from e-cigarettes was collected using a Cerulean CETI-8
aerosol collection system directly into two serial glass impingers, each
containing 35 mL of 6 mM DNPH·HCl solution in acetonitrile/DI water
1:1 (no additional phosphoric acid needed vs CRM #74).
• One port of the CETI-8 was used for air blank sample.
• After aerosol collection, the solutions containing derivatized carbonyls
in two serial impingers were combined and an aliquot of 7 mL
transferred to a scintillation vial.
• 0.15 mL of 3.9 % Trizma base aqueous solution was added to the vial
to neutralize the solution.
• Using a 0.2 µm polypropylene filter, an aliquot was transferred into an
autosampler vial for analysis by UPLC-MS (autosampler temperature:
set at 5 °C).
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6. Optimization – Chromatography
Use of ammonium acetate as additive.
Optimal condition: 1 mM ammonium acetate.
Standard S/N Formaldehyde Acetaldehyde Acrolein Crotonaldehyde
Std 1S (1ng/mL)* 21.9 27.7 21.3 66.4
Std 1(2ng/mL) 49.2 70.4 77.9 90.6
*: used to quantitate results for samples spiked at standard level 1 as LOQ (2 ng/mL) during the validation – the target range for most analytes.
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7. Expand Calibration Range
➢ Challenge: response of the detector is not linear at lower concentrations
versus higher concentrations.
➢ Solution: use of dual calibration curves. Each standard level injected
once in the same run.
Standard Level Concentration Curve 1 Curve 2
Level 1 2 ng/mL √
Level 2 4 ng/mL √
Level 3 10 ng/mL √ √
Level 4 20 ng/mL √ √
Level 5 40 ng/mL √ √
Level 6 100 ng/mL √ √
Level 7 200 ng/mL √
Level 8 400 ng/mL √
R2
Formaldehyde Acetaldehyde Acrolein Crotonaldehyde Curve Fit Weighting
Curve 1 0.99985 0.99985 0.99930 0.99968 quadratic 1/x
Curve 2 0.99928 0.99949 0.99986 0.99982 quadratic (or linear) 1/x
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8. LC-MS Conditions
Waters ACQUITY UPLC H Class with QDa Performance detector
• Column: Waters ACQUITY UPLC BEH Shield RP18 1.7 µm 2.1x100 mm
• Column temperature: 50 °C; Flow rate: 0.6 mL/min
• Injection volume: 1 µL
• Mobile phase A: 1 mM aqueous ammonium acetate: acetonitrile 4:1
• Mobile phase B: acetonitrile
Time (min) Mobile A (%) Mobile B (%) Curve
0 93 7 6
5 93 7 6
7 63 37 6
9.1 63 37 6
9.2 25 75 6
10.4 25 75 6
10.5 93 7 6
12 93 7 6
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9. LC-MS Conditions (Continued)
Compound Mass (Da)
Cone Voltage
(V)
Time Segment
(min)
Formaldehyde-DNPH 209 2 3.8 – 6
Acetaldehyde-DNPH 223 2 6 – 7.3
Acrolein-DNPH 235 2 7.3 – 8.3
Crotonaldehyde-DNPH 249 2 8.3 – 9.5
• Source temperature: 150 °C
• Probe temperature: 600 °C
• Capillary: 0.8 kV
• Ionization mode: electrospray
• Polarity: negative
• Divert to waste: before 3.8 min and after 9.5 min
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10. Results – Chromatograms
- Commercial e-cig aerosol sample spiked at 2 ng/mL
Formaldehyde Acetaldehyde
Acrolein Crotonaldehyde
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11. Recoveries for E-Vapor Product Samples
%Recovery %RSD %Recovery %RSD %Recovery %RSD %Recovery %RSD
LOQ Spike 114.2% 3.2% 108.0% 2.0% 106.5% 2.2% 101.7% 2.5%
Low Spike - Curve 1 101.9% 1.6% 99.4% 1.4% 94.1% 0.7% 100.8% 1.5%
Mid Spike - Curve 1 96.1% 0.7% 99.5% 0.7% 97.1% 1.4% 97.8% 0.8%
High Spike - Curve 1 100.4% 2.2% 98.9% 3.3% 99.9% 2.0% 98.2% 2.1%
Low Spike - Curve 2 101.9% 0.6% 99.4% 0.6% 94.1% 1.3% 100.8% 0.7%
Mid Spike - Curve 2 96.1% 2.4% 99.5% 3.6% 97.1% 2.1% 97.8% 2.3%
High Spike - Curve 2 100.4% 1.4% 98.9% 1.5% 99.9% 1.4% 98.2% 1.2%
Formaldehyde Acetaldehyde Acrolein Crotonaldehyde
Aerosol
ISO 20768:2018
%Recovery %RSD %Recovery %RSD %Recovery %RSD %Recovery %RSD
LOQ Spike 102.4% 1.8% 97.8% 0.8% 95.7% 0.7% 92.7% 1.9%
Low Spike - Curve 1 110.8% 0.5% 80.5% 2.7% 92.4% 1.5% 90.2% 1.2%
Mid Spike - Curve 1 111.8% 1.1% 93.3% 1.5% 94.0% 2.7% 91.7% 1.4%
High Spike - Curve 1 106.9% 0.7% 100.5% 0.9% 99.0% 1.3% 98.4% 1.6%
Low Spike - Curve 2 96.1% 1.1% 93.6% 1.4% 94.7% 2.5% 93.2% 1.3%
Mid Spike - Curve 2 103.6% 0.8% 98.8% 0.9% 97.7% 1.4% 96.7% 1.7%
High Spike - Curve 2 104.6% 0.9% 99.7% 0.5% 98.1% 0.7% 97.6% 1.5%
Formaldehyde Acetaldehyde Acrolein Crotonaldehyde
E-Liquid
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12. Conclusions
• This method provides a simplified solution for the analysis of mono-carbonyls at
trace or non-detectable levels in complex e-vapor product sample matrices with
good sensitivity and selectivity by UPLC-MS.
• The method development process involved the optimization of the derivatization
process such as pH and DNPH concentrations, chromatography conditions as
well as the mass spec detector settings and its linear response range.
• The scope of our method has been expanded to include e-liquid samples.
• The method may be applicable to smokeless and heated tobacco product
analyses, which also have trace or non-detectable levels of mono-carbonyl
compounds.
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